Process for the manufacture of cardioactive monoglycosides



' 3 177 200 i PROCESS FOR THE MANI JFACTURE F CARDIU- ACTIVE MUNQGLYCOSEDES Kuno Meyer, Basel, Switzerland, assignor to Hofimanm La Roche Inc., Nutley, N.J., a corporation of New Jersey No Drawing. Filed July 31, 1963, Ser. No. 299,1ti3 Claims priority, application Switzeriand, Aug. Ill), 1962,

9,607/62 2 Claims. ((Il. 266-4165) The present invention relates to cardioactive monoglycosides, and more particularly, to cardioactive monoglycosides and to a process for their preparation.

The process of the invention is carried out by fermentatively degrading the diglycosides extracted from Digitalis canariensis L. in the form of a mixture to the corresponding monosides and, if desired, separating the resulting monoside mixture into its components.

Digitalis canariensis L. (Canary foxglove) is a kind of Digitalis indigenous to the Canary Islands. with the Digitalis isabelliana (Digitalis canariensis L. var. isabelliana), which is only found in the Grand Canary Island, it forms the subgenus Isoplexis which distinguishes itself from the other herbal kinds of Digitalis (subgenus Eudigitalis) by its shrub-like growth and by evergreen leaves.

Digitalis canariensis L. is characterized by a high content of cardiac glycosides. times higher than with Digitalis pm'purea.

It has now been discovered that the predominant gly- Together This content is, on average,

coside of Digitalis canariensis L. is based upon a hitherto .unknown aglycone which has been named canarigenin and has the following formula In this predominant glycoside, the above aglycone '[A 65,14-dihydroxy-card-2O(22)-enolide] is linked with digitoxose which, in turn, is bound to glucose.

The glycoside mixture used as a starting material in the practice of the process of the invention can be ob- 'tained by known methods; for example, in accordance I with the extraction process described in Helv. Chim. Acta,

1959, 42, 1014. This extraction process can be carried out by'exhaustively extracting the drug-containing material, especially the dried leaves of Digitalis canariensis L. with an aqueous alkanol, eg., methanol or ethanol, and/ or an anhydrous alkanol, precipitating the accompanying materials from the extract obtained by means of basic lead acetate; and finally undertaking a distribution of the extract between the aqueous phase and dilierent organic solventsfor example, in the sequence ether, chloroform, chloroform/ethanol (4:1), chloroform/ ethanol (2:1) and chloroform/ethanol (3:2). .133 this procedure most of the cardioactivecontent passes into the chloroform/ethanol (4:1) extract. The glycoside mixture contained in represents a preferred starting material. However, the glycoside mixture which 'is found in the aqueous phase after theextraction with'ether and chloroform only can also be used as the starting material;

this extract accordingly- 2 The fermentative degradation of the diglycosides (biosides) contained in the glycoside mixture to the corresponding monoglycosides (monosides) is carried out by the use of enzymes which effect the splitting off of the terminal glucose from the. bioside molecule. Such enzymes are, by way of example, digilanidase (from Digitalis lanata), digipurpidase (from Digitalis purpurea), scillarenase (from Scilla maritima), takadiastase (from Aspergillus oryzae), glucosidases from yeast or from hepatopancreas juice, etc. Generally, the fermentation is elfected at a pH of about 6, at a temperature in the range'oi about 35 to 40 C. and in the presence of a water-miscible solvent.

Strophanthobiase (see, for example, Enzymologia, 1939, 7, 362) is an excellent ferment for use in the practice of the invention. About 0.5 to about 2 parts of strophanthobiase are conveniently used to each part of glycoside mixture. The fermentative degradation can be undertaken in aqueous solution, if desired, with the addition of a water-miscible solvent, for example, a ketone (such as acetone), an ether (such as dioxan,

methyl Cellosolve), a lower alkanol (such as methanol,

ethanol), etc. The duration of the fermentation is in the range of about 5 to about 20 days at a temperature of about 37 C. Moreover, it is to be noted that certain ferments (such as, for example, strophanthobiase) are capable of splitting not only the terminal glucose residue but also the sugar component/which is directly attached to the aglycone. If desired, non-fermented material obtained can be subjected to a refermentation, but, in general, this is not worthwhile. The monosides formed by the fermentative degradation can be separated from unchanged starting material by removing them by shaking with a suitable solvent, for example, a halogenated aliphatic hydrocarbon such as chloroform, methylene chloride, etc. I

As has been found, the canarigenin digitoxoside [sugar component: D(+)-digitoxose] predominates in the monoside mixture obtained.

This monoside has the following formula Other monosides presentin the monoside mixture are canarigenin canaroside [sugar component: D(+)-canarose (2-desoxy-D-rhamnose)] having the formula EH OHYH and xysmalogenin canaroside.

form/methanol as the eluents.

D(+)-canarose-is obtained from canarigenin canaroside by acid hydrolysis, e.g. byv treatment with a warm solutionof methanol/0. 1 N sulfuric acid (1:1).

The separation. of the obtained monoside mixture for the purpose of obtainingthe individual monosides, especially the canarigenin-digitoxoside just mentioned, can be carried out, for example, by fractional crystallization,

countercurrent distribution or, preferably, by chromatographyv (on A1 0 silieagel, etc.) using, for example, benzene/chloroform mixtures, chloroform and chloro- The monosides obtainable according to the process of the invention possess a strong cardiac action, and are useful as cardiac stimulants. They are particularly useful due. to their low cumulationandlow reabsorbability. Canarigenin is useful in the preparation of the above designated canarigenin monosides.

EXAMPLE 1 1 (a)Fermentation-84g. ofa powdered glycoside mixture [obtained from leaves of Digitalis canariensis L. in the form of the chloroform/ethanol (4:1) extract] are dissolved in 1200ml. of methanol/water(1:1) with gentle heating. A total of 600 ml. of methanol and 18 liters of warm water are added gradually and'alternately to p the honey-brown clear solution while rotating. The slightly opaque solution thus. obtained is freed from methanol ina thin-layer evaporator at in portions eachofaround 250 ml. To the resulting turbid solution (volume12 liters) is added, while stirring,'a solution of g. of crude. strophanthobiase in'lOOO'mI. of

water (dark-brown colored, opaque) is poured in in a fine jet. After the addition of 10-15 ml. of toluene, the fermentation is'left to stand for 10 days at 37 with I g. of a yellow-white foam are obtained as the residue.

. This is partitioned between 25 ml. of water and 250 ml.

After decanting of chloroform in "a separatory funnel. off the chloroform, the aqueous phase is extracted a further 3 times, each time with 150ml. of chloroform.

in succession through. 2 separating funnels" each-containing 200 ml. of waterand are then worked up as described above. 23.7 g. .of a monosidemixtureare obtained. Thetotal quantity amountstto 28.5 g. (together with the monoside mixture isolated from they fermentation precipitates).

From the fermentation solution extracted with chloroform. as previously described a'total of 32 g. of unfermented material can be recovered by extraction witha chloroform/ethanol (4:1) mixture: Refermentation is not worthwhile. 7

([2) Chromatographic separation of the monos ide mixture-Q8 g. of the monoside mixture obtained are chro-. matographed on 600 g.- of A1 0 The following table gives information concerning the procedure and the results achieved:

Table Isolated substance Fraction number Solvent-(500 ml.) for Composieach fraction Amount tion (crude) (thin-layer in mg. chromatography) Benzene-Chlf. (4:1)"... Benzene-Chlf. (3:2)-.. 160 .Benzene-Chlt'. (3:2)..... 150 a.

Benzene-Chit. (3:7)"--- 195 a, b, c Benzene-Ohlf. (3:7)...-. 655 b, 0 13-15 chloroform 1,100 b, c, (d) 16-17.-- .do 1,030 b, (c), (I 18-35 .-do 10, 445 d '36-40 Ghlt-Me (99.5:0,5)---.. 645 d, e

Chit-Me (991).; 1, 605 e,]', g Chit-Me (49:1) 2,030 e, f, g Chlt-He (19:1)... 4, 600 e, f, g ChlL-Me (9:1).... 435 c,f, g Chit-Me (7:3) e, f; g

. ChlLzChl'oroiorm.

Me:Methan0l. a Canarigenin 3-methyl ether.- b Canarigenin. Y c:3-ep1canarigenin. I d:Canarigenin-digitoxoside. 7 czMixtureotmonosides consisting of canarigenin cauaroside and xysmalogenin eanaroside. I v

f, gzNon-identified.monosides.

7.75 g. of pure canarigenin-digitoxoside and a further 1.3

g. of canarigenin-digitoxoside containing some monosides are obtained from fractions18-35.

Properties of the .pure canarig enjndigitoxoside: finev small needles (containing water) of melting point 176-201 F C. from acetone/ether; prismatic plates (solvent-free) of The yellow filtrateis brought to dryness in a vacuum. 5.6 50

After washing with water, drying over sodium sulfate and evaporating in a vacuum, the chloroform extractsgive a total of. 4.8 g. of monosidemixture. The aqueous solution (25 ml.) is extracted 3 times, each time with 150 ml..of chloroform/ethanol (2:1), 0.8 g. of starting mate-- rial are thus recovered. V

The, fermentation solution freed from the'precipitate pressure at 50 and treated with 12 liters of 96 percent ethanol, heated to boiling on they steam-bath and theretreated with 1500 ml. of water and concentrated to 1000 ml. in a rotation evaporator (strong foaming).

out. The solution and precipitate are extracted in a 3- ;liter separating funnel 10 times with 1000 ml. of chloroform each'time; The chloroformv extracts are then passed (see above) is concentrated m2 liters under reduced 1 melting point 142 C. (frommethanol/ether);

(in chloroform). The acetyl compound obtainable in the form of very fine small-needles, melts-at 212-225 C. (fromacetone); [a] ;+4.2:-2 (in chloroform).

If the.canarigenin-digitoxoside.is subjected to hydrolysis according to methods commonly used for the splitting of Z-desoxy-sugar glycosides [heating on the steam bath for 30 minutes in methanol/(0.1-N sulfuric acid) (1:1)] then one molecule of water'is eliminatedifrom the genuine- .aglycone (canarigenin) and the 3,S-dianhydro-periplogenin is obtained as the aglycone. 1

The splitting of canarigenin-di-gitoxoside' with methanol/ water and 1 percent glacial acetic (37 C., left standing ;for 8-10 days) yie1ds,"in' the main, the canarigenin B-methyl ether of melting point, 212-222" C The splitting of 'canarigenin-digitoxosideywith ,Vacetone/water (1:1) and 1 percent glacialacetic (37 0., left standing for 8-10 days) yields, as the main-product, canar- (in chloroform) and ,3,5:dianhydro-periplogenin... I

the formation of 3-epi-ca1narigenin it follows that inversion at C of the aglycone can occur with splitting.

The aglycone, canarigenin, forms prismatic plates from chloroform/methanol/acetone. Melting point 235252 C.; [cc] =+22i2 (in methanol). The acetyl-canarigenin melts at 214 217" 0; [a] =4 12 (in chloroform). Cautious dehydrogenation of canarigenin leads to anhydro-periplogenone.

The product designated as e (see the above table), present in fractions 3647, is a chromatographically uniform mixture of two isomeric monosides both containing the same sugar component, viz. D(+)-canarose(2-desoxy- D-rharnnose). The aglycones are canarigenin and xysmalogenin, respectively. Accordingly, the product designated as "e is a mixture of canarigenin-D(-|-)-canaroside and xysmalogenin-D(,+)-canaroside. The mixture has the double melting point 150-155 /218235 (from methanol), [a] =3l.8i2 (in chloroform). Melting point of the acetyl derivative of the monoside mixture 265270 C.; [a] =-21 i2 (in chloroform).

(c) Obtaining the starting material.-1200 g. of airdried leaves of Digitalis canariensis L., which have been ground as fine as dust, in a ball-mill, are well worked through with 2 liters of warm water, thereafter treated with 2 liters of ethanol at 60 C. Then the whole is fil tered while still warm over a filter packed with Hyflo Supercel and the residue is extracted in the same manner yet 6 times, each time with 2 liters of ethanol. The combined extracts are concentrated in a vacuum at 50 (bath temperature) to a total volume of about 1.2 liters. After the addition of about 1.2 liters of water, the about 50 percent ethanol solution is shaken on a machine for 30 minutes with Pb(OI-l) (freshly prepared from 1.2 kg. of lead diacetate trihydrate and Washed until neutral), filtered under suction through a filter packed with Hyfio Supercel, the residue made into a paste with 500 ml. of 50 percent ethanol, again filtered off under suction and rewashed with a little 50 percent ethanol. The filtrate only just gives a slight turbidity with aqueous lead acetate solution. It is adjused to pH 6 with 2-N sulfuric acid and concentrated to 1 liter while maintaining a pH of about 6. This solution is extracted in a separating funnel 4 times, each time with 2 liters of ether, then 4 times, each time with 1 liter of chloroform, 8 times, each time with 1 liter of chloroform/ ethanol (4:1),3 times, each time with 1 liter of chloroform/ ethanol (2:1) and finally a further 3 times, each time with 1 liter of chloroform/ ethanol (3:2). All extracts are passed in succession through two shaking funnels, each containing 150 ml. of water (the ether extracts alone are additionally washed with dilute soda solution) and are dried over sodium sulfate, then filtered and evaporated under vacuum.

On an average, there are obtained from 1 kg. of leafpowder the following quantities of material:

The glycoside mixture extracted with chloroform/ethanol (421) is used as the starting material.

I claim:

1. Canarigenin-D(+)-digitoxoside having the formula 2. Canarigenin, having the formula References Cited by the Examiner UNITED STATES PATENTS 2,179,204 11/39 Stoll et al 260-2105 2,752,372 6/ 56 Reichstein 260-2105 OTHER REFERENCES Studer et al.: Helv. Chem. Acta., vol. XLVI, Feb. 1, 1963, pp. 23-45.

LEWIS GOTTS, Primary Examiner.

iLc/O 

1. CANARIGENIN-D(+)-DIGITOXOSIDE HAVING THE FORMULA 